Study population and setting
A full length complimentary DNA (cDNA) clone of SARS-CoV-2 was assembled from 7 cDNA fragments based on the virus strain 2019-nCoV/USA_WA1/2020, which was isolated from the first reported SARS-CoV-2 case in the US. cDNA can be used to clone genes for expression of proteins. Additionally, standard molecular cloning techniques were used to introduce a reporter gene (mNeonGreen) into ORF7 of the viral genome. A reporter gene is a gene that is attached to a regulatory sequence of a gene of interest such that the gene of interest can easily be identified and measured. Viral replication kinetics were measured as well as stability of the reporter gene in serial passages.
Summary of Main Findings
The wild-type virus was able to be produced from a reverse genetics system. Genome fidelity to the wild-type was confirmed by sequencing. This virus has replication kinetics and mRNA expression similar to that of the wild-type virus, and displays similar type-1 interferon sensitivity as wild type. Addition of the mNeonGreen reporter gene did not attenuate replication. The reporter gene was stable up to at least 5 passages in cell culture.
Development of a reverse genetics system for SARS-CoV-2 is essential to allow future robust molecular biology studies to be performed. The authors included three engineered synonymous mutations into the viral cDNA which allow for distinguishing from wild-type virus by sequencing. Replication kinetics of both the wild-type molecular clone and the mNeonGreen reporter virus were measured and were similar to that of wild-type virus. This is an extremely important characteristic to ensure that these viruses are valid for use in future studies.
All molecular clones carry the limitation that they are only representative of the original virus from which they were derived. As the SARS-CoV-2 virus is still new in the population and still rapidly circulating, it remains to be seen if major major mutations will arise in the genome that will need to be represented in any molecular clones used for further study.
The development of a reverse genetics system and reporter virus for SARS-CoV-2 is essential to allow future robust molecular biology studies to be performed. This will significantly advance our ability to characterize this virus, as well as construct attenuated clones that may be useful as vaccines.